Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Methodological advances: the unsung heroes of the GPCR structural revolution

Nature Reviews Molecular Cell Biology volume 16, pages 69–81 (2015)Cite this article

Subjects

Key Points

  • G protein-coupled receptors (GPCRs) constitute the largest family of cell-surface receptors and they are the primary targets of approximately half of the currently prescribed drugs.

  • A range of methodological advances were necessary to crystallize GPCRs and to determine their three-dimensional crystal structures.

  • Protein engineering, new detergents, synthetic crystallization chaperones, novel crystallization strategies and microfocus synchrotron beamlines were pivotal to the successful generation of GPCR crystals and the determination of their structures.

  • A crystal structure of a prototypical GPCR, the β2-adrenoceptor, has been determined in a complex with its primary signalling effector, the heterotrimeric G protein.

  • Future studies focused on the structural basis of GPCR–effector interactions and biased signalling conformations should provide the missing link to develop a more complete understanding of the mechanistic basis of GPCR activation and signalling.

  • High-resolution visualization of the ligand-binding pocket of GPCRs provides a framework for structure-based novel drug discovery to target GPCRs that are involved in the pathogenesis of many human diseases.

Abstract

G protein-coupled receptors (GPCRs) are intricately involved in a diverse array of physiological processes and pathophysiological conditions. They constitute the largest class of drug target in the human genome, which highlights the importance of understanding the molecular basis of their activation, downstream signalling and regulation. In the past few years, considerable progress has been made in our ability to visualize GPCRs and their signalling complexes at the structural level. This is due to a series of methodological developments, improvements in technology and the use of highly innovative approaches, such as protein engineering, new detergents, lipidic cubic phase-based crystallization and microfocus synchrotron beamlines. These advances suggest that an unprecedented amount of structural information will become available in the field of GPCR biology in the coming years.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A simplified schematic of GPCR signalling.
Figure 2: Overview of GPCR crystallography, its major challenges and methodological solutions to these challenges.
Figure 3: Protein engineering approaches to stabilize the third intracellular loop of GPCRs for crystallography.
Figure 4: Protein engineering approaches to stabilize GPCRs outside the third intracellular loop for crystallography.
Figure 5: Schematic representation of conventional vapour diffusion, bicelle and lipid cubic phase crystallization approaches.
Figure 6: Trapping active GPCR conformations using nanobody technology.
Figure 7: Visualizing GPCR signalling complexes.

Similar content being viewed by others

References

  1. Pierce, K. L., Premont, R. T. & Lefkowitz, R. J. Seven-transmembrane receptors. Nature Rev. Mol. Cell Biol. 3, 639–650 (2002).

    Article  CAS  Google Scholar 

  2. Bockaert, J. & Pin, J. P. Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J. 18, 1723–1729 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Lagerstrom, M. C. & Schioth, H. B. Structural diversity of G protein-coupled receptors and significance for drug discovery. Nature Rev. Drug Discov. 7, 339–357 (2008).

    Google Scholar 

  4. Foord, S. M. et al. International Union of Pharmacology. XLVI. G protein-coupled receptor list. Pharmacol. Rev. 57, 279–288 (2005).

    CAS  PubMed  Google Scholar 

  5. Conn, P. M., Ulloa-Aguirre, A., Ito, J. & Janovick, J. A. G protein-coupled receptor trafficking in health and disease: lessons learned to prepare for therapeutic mutant rescue in vivo. Pharmacol. Rev. 59, 225–250 (2007).

    CAS  PubMed  Google Scholar 

  6. Ma, P. & Zemmel, R. Value of novelty? Nature Rev. Drug Discov. 1, 571–572 (2002).

    CAS  Google Scholar 

  7. Zalewska, M., Siara, M. & Sajewicz, W. G protein-coupled receptors: abnormalities in signal transmission, disease states and pharmacotherapy. Acta Poloniae Pharmaceut. 71, 229–243 (2014).

    Google Scholar 

  8. Reiter, E., Ahn, S., Shukla, A. K. & Lefkowitz, R. J. Molecular mechanism of β-arrestin-biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52, 179–197 (2012).

    CAS  PubMed  Google Scholar 

  9. Reiter, E. & Lefkowitz, R. J. GRKs and β-arrestins: roles in receptor silencing, trafficking and signaling. Trends Endocrinol. Metabolism 17, 159–165 (2006).

    CAS  Google Scholar 

  10. Deupi, X., Standfuss, J. & Schertler, G. Conserved activation pathways in G-protein-coupled receptors. Biochem. Soc. Trans. 40, 383–388 (2012).

    CAS  PubMed  Google Scholar 

  11. Kang, D. S., Tian, X. & Benovic, J. L. Role of β-arrestins and arrestin domain-containing proteins in G protein-coupled receptor trafficking. Curr. Opin. Cell Biol. 27, 63–71 (2014).

    PubMed  Google Scholar 

  12. Tian, X., Kang, D. S. & Benovic, J. L. β-arrestins and G protein-coupled receptor trafficking. Handb. Exp. Pharmacol. 219, 173–186 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Hall, R. A., Premont, R. T. & Lefkowitz, R. J. Heptahelical receptor signaling: beyond the G protein paradigm. J. Cell Biol. 145, 927–932 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Lefkowitz, R. J. Seven transmembrane receptors: something old, something new. Acta Physiol. 190, 9–19 (2007).

    CAS  Google Scholar 

  15. Goodman, O. B. Jr. et al. β-arrestin acts as a clathrin adaptor in endocytosis of the β2-adrenergic receptor. Nature 383, 447–450 (1996).

    CAS  PubMed  Google Scholar 

  16. Shukla, A. K., Xiao, K. & Lefkowitz, R. J. Emerging paradigms of β-arrestin-dependent seven transmembrane receptor signaling. Trends Biochem. Sci. 36, 457–469 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Shukla, A. K. Biasing GPCR signaling from inside. Sci. Signal. 7, pe3 (2014).

    PubMed  Google Scholar 

  18. DeWire, S. M., Ahn, S., Lefkowitz, R. J. & Shenoy, S. K. β-arrestins and cell signaling. Annu. Rev. Physiol. 69, 483–510 (2007).

    CAS  PubMed  Google Scholar 

  19. Rasmussen, S. G. et al. Crystal structure of the human β2 adrenergic G-protein-coupled receptor. Nature 450, 383–387 (2007). This paper describes the structure of the human β 2 -adrenoceptor, the first non-rhodopsin GPCR crystal structure.

    CAS  PubMed  Google Scholar 

  20. Katritch, V., Cherezov, V. & Stevens, R. C. Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013).

    CAS  PubMed  Google Scholar 

  21. Rasmussen, S. G. et al. Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549–555 (2011). This paper presents the crystal structure of a human β 2 -adrenoceptor–G protein complex.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Shukla, A. K. et al. Visualization of arrestin recruitment by a G-protein-coupled receptor. Nature 512, 218–222 (2014). This paper describes the low-resolution architecture of a GPCR–β-arrestin complex and directly visualizes the biphasic mechanism of the GPCR-β-arrestin interaction.

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Shukla, A. K. et al. Structure of active β-arrestin-1 bound to a G-protein-coupled receptor phosphopeptide. Nature 497, 137–141 (2013). This paper describes the crystal structure of activated β-arrestin 1 in complex with the phosphorylated C terminus of a GPCR.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Lundstrom, K. et al. Structural genomics on membrane proteins: comparison of more than 100 GPCRs in 3 expression systems. J. Struct. Funct. Genom. 7, 77–91 (2006).

    CAS  Google Scholar 

  25. Roy, A., Shukla, A. K., Haase, W. & Michel, H. Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors. Biol. Chem. 389, 69–78 (2008).

    CAS  PubMed  Google Scholar 

  26. Shukla, A. K., Haase, W., Reinhart, C. & Michel, H. Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells. Int. J. Biochem. Cell Biol. 39, 931–942 (2007).

    CAS  PubMed  Google Scholar 

  27. Shukla, A. K., Haase, W., Reinhart, C. & Michel, H. Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris. Protein Expression Purif. 55, 1–8 (2007).

    CAS  Google Scholar 

  28. Shukla, A. K., Haase, W., Reinhart, C. & Michel, H. Functional overexpression and characterization of human bradykinin subtype 2 receptor in insect cells using the baculovirus system. J. Cell. Biochem. 99, 868–877 (2006).

    CAS  PubMed  Google Scholar 

  29. Shukla, A. K., Reinhart, C. & Michel, H. Dimethylsulphoxide as a tool to increase functional expression of heterologously produced GPCRs in mammalian cells. FEBS Lett. 580, 4261–4265 (2006).

    CAS  PubMed  Google Scholar 

  30. Shukla, A. K., Haase, W., Reinhart, C. & Michel, H. Biochemical and pharmacological characterization of the human bradykinin subtype 2 receptor produced in mammalian cells using the Semliki Forest virus system. Biol. Chem. 387, 569–576 (2006).

    CAS  PubMed  Google Scholar 

  31. Shukla, A. K., Reinhart, C. & Michel, H. Comparative analysis of the human angiotensin II type 1a receptor heterologously produced in insect cells and mammalian cells. Biochem. Biophys. Res. Commun. 349, 6–14 (2006).

    CAS  PubMed  Google Scholar 

  32. Cherezov, V. et al. High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318, 1258–1265 (2007). This paper describes the first high-resolution structure of a non-rhodopsin GPCR: the human β 2 -adrenoceptor.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Shimamura, T. et al. Structure of the human histamine H1 receptor complex with doxepin. Nature 475, 65–70 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Egloff, P. et al. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl Acad. Sci. USA 111, E655–E662 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Chelikani, P., Reeves, P. J., Rajbhandary, U. L. & Khorana, H. G. The synthesis and high-level expression of a β2-adrenergic receptor gene in a tetracycline-inducible stable mammalian cell line. Protein Sci.: Publ. Protein Soc. 15, 1433–1440 (2006).

    CAS  Google Scholar 

  36. Cook, B. L., Ernberg, K. E., Chung, H. & Zhang, S. Study of a synthetic human olfactory receptor 17-4: expression and purification from an inducible mammalian cell line. PloS one 3, e2920 (2008).

    PubMed  PubMed Central  Google Scholar 

  37. Wang, X. & Zhang, S. Production of a bioengineered G-protein coupled receptor of human formyl peptide receptor 3. PloS one 6, e23076 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Standfuss, J. et al. Crystal structure of a thermally stable rhodopsin mutant. J. Mol. Biol. 372, 1179–1188 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Andre, N. et al. Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen. Protein Sci. 15, 1115–1126 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kobilka, B. K. Amino and carboxyl terminal modifications to facilitate the production and purification of a G protein-coupled receptor. Anal. Biochem. 231, 269–271 (1995).

    CAS  PubMed  Google Scholar 

  41. Gether, U. et al. Agonists induce conformational changes in transmembrane domains III and VI of the β2 adrenoceptor. EMBO J. 16, 6737–6747 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kobilka, B. K. & Gether, U. Use of fluorescence spectroscopy to study conformational changes in the β2-adrenoceptor. Methods Enzymol. 343, 170–182 (2002).

    PubMed  Google Scholar 

  43. Day, P. W. et al. A monoclonal antibody for G protein-coupled receptor crystallography. Nature Methods 4, 927–929 (2007). This paper describes the generation of a monoclonal antibody against the human β 2 -adrenoceptor, which was successfully used to obtain crystals of the first non-rhodopsin GPCR.

    CAS  PubMed  Google Scholar 

  44. Ostermeier, C., Iwata, S., Ludwig, B. & Michel, H. Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase. Nature Struct. Biol. 2, 842–846 (1995).

    CAS  PubMed  Google Scholar 

  45. Hunte, C., Koepke, J., Lange, C., Rossmanith, T. & Michel, H. Structure at 2.3 A resolution of the cytochrome bc(1) complex from the yeast Saccharomyces cerevisiae co-crystallized with an antibody Fv fragment. Structure 8, 669–684 (2000).

    CAS  PubMed  Google Scholar 

  46. Padan, E., Venturi, M., Michel, H. & Hunte, C. Production and characterization of monoclonal antibodies directed against native epitopes of NhaA, the Na+/H+ antiporter of Escherichia coli. FEBS Lett. 441, 53–58 (1998).

    CAS  PubMed  Google Scholar 

  47. Hunte, C. & Michel, H. Crystallisation of membrane proteins mediated by antibody fragments. Curr. Opin. Struct. Biol. 12, 503–508 (2002).

    CAS  PubMed  Google Scholar 

  48. Screpanti, E., Padan, E., Rimon, A., Michel, H. & Hunte, C. Crucial steps in the structure determination of the Na+/H+ antiporter NhaA in its native conformation. J. Mol. Biol. 362, 192–202 (2006).

    CAS  PubMed  Google Scholar 

  49. Hino, T. et al. G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody. Nature 482, 237–240 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Rosenbaum, D. M. et al. GPCR engineering yields high-resolution structural insights into β2-adrenergic receptor function. Science 318, 1266–1273 (2007). This paper describes the T4L fusion-based protein engineering approach used for crystallizing the human β 2 -adrenoceptor.

    CAS  PubMed  Google Scholar 

  51. Haga, K. et al. Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist. Nature 482, 547–551 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Kruse, A. C. et al. Structure and dynamics of the M3 muscarinic acetylcholine receptor. Nature 482, 552–556 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Manglik, A. et al. Crystal structure of the micro-opioid receptor bound to a morphinan antagonist. Nature 485, 321–326 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Zhang, C. et al. High-resolution crystal structure of human protease-activated receptor 1. Nature 492, 387–392 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Granier, S. et al. Structure of the delta-opioid receptor bound to naltrindole. Nature 485, 400–404 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Jaakola, V. P. et al. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science 322, 1211–1217 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Chien, E. Y. et al. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 330, 1091–1095 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Wu, B. et al. Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science 330, 1066–1071 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Thompson, A. A. et al. Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic. Nature 485, 395–399 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Wu, H. et al. Structure of the human κ-opioid receptor in complex with JDTic. Nature 485, 327–332 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Siu, F. Y. et al. Structure of the human glucagon class B G-protein-coupled receptor. Nature 499, 444–449 (2013).

    CAS  PubMed  Google Scholar 

  62. Dore, A. S. et al. Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature 511, 557–562 (2014).

    CAS  PubMed  Google Scholar 

  63. Hollenstein, K. et al. Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature 499, 438–443 (2013).

    CAS  PubMed  Google Scholar 

  64. Hanson, M. A. et al. Crystal structure of a lipid G protein-coupled receptor. Science 335, 851–855 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Chun, E. et al. Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure 20, 967–976 (2012). This study tests a number of non-T4L fusion partners for their ability to facilitate the crystallization of GPCRs.

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions. Science 337, 232–236 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Tan, Q. et al. Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science 341, 1387–1390 (2013).

    CAS  PubMed  Google Scholar 

  68. Zou, Y., Weis, W. I. & Kobilka, B. K. N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor. PloS one 7, e46039 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Warne, T., Serrano-Vega, M. J., Tate, C. G. & Schertler, G. F. Development and crystallization of a minimal thermostabilised G protein-coupled receptor. Protein Expression Purif. 65, 204–213 (2009).

    CAS  Google Scholar 

  70. Warne, T. et al. Structure of a β1-adrenergic G-protein-coupled receptor. Nature 454, 486–491 (2008). This paper describes the crystal structure of a β 1 -adrenoceptor obtained using a thermostabilization strategy.

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Tate, C. G. & Schertler, G. F. Engineering G protein-coupled receptors to facilitate their structure determination. Curr. Opin. Struct. Biol. 19, 386–395 (2009).

    CAS  PubMed  Google Scholar 

  72. Dore, A. S. et al. Structure of the adenosine A2A receptor in complex with ZM241385 and the xanthines XAC and caffeine. Structure 19, 1283–1293 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Srivastava, A. et al. High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature 513, 124–127 (2014).

    CAS  PubMed  Google Scholar 

  74. White, J. F. et al. Structure of the agonist-bound neurotensin receptor. Nature 490, 508–513 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Hanson, M. A. et al. A specific cholesterol binding site is established by the 2.8 A structure of the human β2-adrenergic receptor. Structure 16, 897–905 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Thompson, A. A. et al. GPCR stabilization using the bicelle-like architecture of mixed sterol-detergent micelles. Methods 55, 310–317 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Chae, P. S. et al. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nature Methods 7, 1003–1008 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Chae, P. S. et al. Glucose-neopentyl glycol (GNG) amphiphiles for membrane protein study. Chem. Commun. 49, 2287–2289 (2013).

    CAS  Google Scholar 

  79. Chae, P. S. et al. Novel tripod amphiphiles for membrane protein analysis. Chemistry 19, 15645–15651 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Lee, S. C. et al. Steroid-based facial amphiphiles for stabilization and crystallization of membrane proteins. Proc. Natl Acad. Sci. USA 110, E1203–E1211 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Tao, H. et al. Synthesis and properties of dodecyl trehaloside detergents for membrane protein studies. Langmuir. 28, 11173–11181 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Caron, M. G., Srinivasan, Y., Pitha, J., Kociolek, K. & Lefkowitz, R. J. Affinity chromatography of the β-adrenergic receptor. J. Biol. Chem. 254, 2923–2927 (1979).

    CAS  PubMed  Google Scholar 

  83. Lefkowitz, R. J., Sun, J. P. & Shukla, A. K. A crystal clear view of the β2-adrenergic receptor. Nature Biotech. 26, 189–191 (2008).

    CAS  Google Scholar 

  84. Shukla, A. K., Sun, J. P. & Lefkowitz, R. J. Crystallizing thinking about the β2-adrenergic receptor. Mol. Pharmacol. 73, 1333–1338 (2008).

    CAS  PubMed  Google Scholar 

  85. Rosenbaum, D. M. et al. Structure and function of an irreversible agonist–β2 adrenoceptor complex. Nature 469, 236–240 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Rasmussen, S. G. et al. Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175–180 (2011). This paper presents the crystal structure of a fully activated β 2 -adrenoceptor stabilized by a nanobody.

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Weichert, D. et al. Covalent agonists for studying G protein-coupled receptor activation. Proc. Natl Acad. Sci. USA 111, 10744–10748 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. Huang, J., Chen, S., Zhang, J. J. & Huang, X. Y. Crystal structure of oligomeric β1-adrenergic G protein-coupled receptors in ligand-free basal state. Nature Struct. Mol. Biol. 20, 419–425 (2013).

    CAS  Google Scholar 

  89. Park, J. H., Scheerer, P., Hofmann, K. P., Choe, H. W. & Ernst, O. P. Crystal structure of the ligand-free G-protein-coupled receptor opsin. Nature 454, 183–187 (2008).

    CAS  PubMed  Google Scholar 

  90. Faham, S. & Bowie, J. U. Bicelle crystallization: a new method for crystallizing membrane proteins yields a monomeric bacteriorhodopsin structure. J. Mol. Biol. 316, 1–6 (2002).

    CAS  PubMed  Google Scholar 

  91. Faham, S. et al. Crystallization of bacteriorhodopsin from bicelle formulations at room temperature. Protein Sci. 14, 836–840 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F. & Caffrey, M. A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases. Acta Crystallogr. D Biol. Crystallogr. 60, 1795–1807 (2004).

    PubMed  Google Scholar 

  93. Misquitta, L. V. et al. Membrane protein crystallization in lipidic mesophases with tailored bilayers. Structure 12, 2113–2124 (2004).

    CAS  PubMed  Google Scholar 

  94. Cherezov, V., Clogston, J., Papiz, M. Z. & Caffrey, M. Room to move: crystallizing membrane proteins in swollen lipidic mesophases. J. Mol. Biol. 357, 1605–1618 (2006).

    CAS  PubMed  Google Scholar 

  95. Caffrey, M. & Cherezov, V. Crystallizing membrane proteins using lipidic mesophases. Nature Protoc. 4, 706–731 (2009).

    CAS  Google Scholar 

  96. Nollert, P., Qiu, H., Caffrey, M., Rosenbusch, J. P. & Landau, E. M. Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases. FEBS Lett. 504, 179–186 (2001).

    CAS  PubMed  Google Scholar 

  97. Rummel, G. et al. Lipidic cubic phases: new matrices for the three-dimensional crystallization of membrane proteins. J. Struct. Biol. 121, 82–91 (1998).

    CAS  PubMed  Google Scholar 

  98. Chiu, M. L. et al. Crystallization in cubo: general applicability to membrane proteins. Acta Crystallogr. D Biol. Crystallogr. 56, 781–784 (2000).

    CAS  PubMed  Google Scholar 

  99. Landau, E. M. & Rosenbusch, J. P. Lipidic cubic phases: a novel concept for the crystallization of membrane proteins. Proc. Natl Acad. Sci. USA 93, 14532–14535 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Pebay-Peyroula, E., Rummel, G., Rosenbusch, J. P. & Landau, E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science 277, 1676–1681 (1997).

    CAS  PubMed  Google Scholar 

  101. Cherezov, V. et al. Rastering strategy for screening and centring of microcrystal samples of human membrane proteins with a sub-10 microm size X-ray synchrotron beam. J. R. Soc. Interface 6, S587–S597 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Cherezov, V., Liu, J., Griffith, M., Hanson, M. A. & Stevens, R. C. LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization. Crystal Growth Design 8, 4307–4315 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Liu, W., Hanson, M. A., Stevens, R. C. & Cherezov, V. LCP-Tm: an assay to measure and understand stability of membrane proteins in a membrane environment. Biophys. J. 98, 1539–1548 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Xu, F., Liu, W., Hanson, M. A., Stevens, R. C. & Cherezov, V. Development of an automated high throughput LCP-FRAP assay to guide membrane protein crystallization in lipid mesophases. Crystal Growth Design 11, 1193–1201 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Chapman, H. N. et al. Femtosecond X-ray protein nanocrystallography. Nature 470, 73–77 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Weierstall, U. et al. Lipidic cubic phase injector facilitates membrane protein serial femtosecond crystallography. Nature Commun. 5, 3309 (2014).

    Google Scholar 

  107. Liu, W. et al. Serial femtosecond crystallography of G protein-coupled receptors. Science 342, 1521–1524 (2013). This paper presents the first example of a GPCR structure determined by xFEL.

    CAS  PubMed  PubMed Central  Google Scholar 

  108. Liu, W., Ishchenko, A. & Cherezov, V. Preparation of microcrystals in lipidic cubic phase for serial femtosecond crystallography. Nature Protoc. 9, 2123–2134 (2014).

    CAS  Google Scholar 

  109. Bokoch, M. P. et al. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor. Nature 463, 108–112 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  110. Yao, X. et al. Coupling ligand structure to specific conformational switches in the β2-adrenoceptor. Nature Chem. Biol. 2, 417–422 (2006).

    CAS  Google Scholar 

  111. Pardon, E. et al. A general protocol for the generation of Nanobodies for structural biology. Nature Protoc. 9, 674–693 (2014).

    CAS  Google Scholar 

  112. Steyaert, J. & Kobilka, B. K. Nanobody stabilization of G protein-coupled receptor conformational states. Curr. Opin. Struct. Biol. 21, 567–572 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. Kruse, A. C. et al. Activation and allosteric modulation of a muscarinic acetylcholine receptor. Nature 504, 101–106 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  114. Ring, A. M. et al. Adrenaline-activated structure of β2-adrenoceptor stabilized by an engineered nanobody. Nature 502, 575–579 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  115. Shukla, A. K. et al. Distinct conformational changes in β-arrestin report biased agonism at seven-transmembrane receptors. Proc. Natl Acad. Sci. USA 105, 9988–9993 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. Westfield, G. H. et al. Structural flexibility of the Gαs α-helical domain in the β2-adrenoceptor Gs complex. Proc. Natl Acad. Sci. USA 108, 16086–16091 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. Chung, K. Y. et al. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor. Nature 477, 611–615 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  118. Gurevich, V. V. & Gurevich, E. V. The structural basis of arrestin-mediated regulation of G-protein-coupled receptors. Pharmacol. Ther. 110, 465–502 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  119. Gurevich, V. V. & Gurevich, E. V. The molecular acrobatics of arrestin activation. Trends Pharmacol. Sci. 25, 105–111 (2004).

    CAS  PubMed  Google Scholar 

  120. Gurevich, V. V. & Gurevich, E. V. Structural determinants of arrestin functions. Progress Mol. Biol. Translat. Sci. 118, 57–92 (2013).

    CAS  Google Scholar 

  121. Szczepek, M. et al. Crystal structure of a common GPCR-binding interface for G protein and arrestin. Nature Commun. 5, 4801 (2014).

    CAS  Google Scholar 

Download references

Acknowledgements

The authors thank the members of the Shukla laboratory for their critical reading of this manuscript. Research in the Shukla laboratory is supported by the Indian Institute of Technology Kanpur, the Department of Science and Technology (India), the Government of India, and the Wellcome Trust/DBT India Alliance.

Author information

Authors and Affiliations

  1. Department of Biological Sciences and Bioengineering, Indian Institute of Technology, 208016, Kanpur, India

    Eshan Ghosh, Punita Kumari, Deepika Jaiman & Arun K. Shukla

Authors
  1. Eshan Ghosh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Punita Kumari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Deepika Jaiman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Arun K. Shukla
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Arun K. Shukla.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

PowerPoint slides

Supplementary information

Supplementary information S1 (table)

A comprehensive list of GPCRs crystallized so far. (XLSX 78 kb)

Related links

Related links

FURTHER INFORMATION

RCSB Protein Data Bank

Glossary

Synchrotron-based X-ray sources

These are powerful X-ray sources that are used to collect high-resolution X-ray diffraction data on three-dimensional crystals. Examples of the synchrotron X-ray sources utilized in G protein-coupled receptor crystallography are the Advanced Photon Source in Chicago (USA) and the European Synchrotron Radiation Facility in Grenoble (France).

Microfocus beamlines

Next-generation X-ray sources at synchrotron facilities that are suitable for the structural analysis of microcrystals (in the size range of 5–20 μm). The most commonly used microfocus beamlines for G protein-coupled receptor crystallography are the ID23 beamline at the European Synchrotron Radiation Facility in Grenoble (France), the I24 beamline at the Diamond Light Source in Oxfordshire (UK) and the 23ID beamline at the Advanced Photon Source in Chicago (USA).

Inverse agonist

Most G protein-coupled receptors when overexpressed display a certain degree of basal or constitutive signalling. Inverse agonists bind to the receptor and reduce its basal or constitutive activity.

Antigen-binding fragment

(Fab). Fab is the region on the antibody that binds antigens and is composed of a heavy chain constant and variable domain, and a light chain constant and variable domain.

Amphiphile

A compound that has both lipophilic and hydrophilic properties.

Lipidic cubic phase

(LCP). A novel crystallization approach in which membrane proteins are embedded in a membrane-mimetic lipid environment for crystallization.

Immobilized metal affinity chromatography

(IMAC). This technique refers to a particular type of affinity chromatography that uses coordinate covalent bond formation between specific amino acids in the protein (most often histidines) and immobilized metal ions (most often Ni2+) on a solid support (for example, agarose beads). Ni-nitrilotriacetic acid resin-based protein purification is one of the most commonly used forms of IMAC.

Ligand affinity chromatography

A purification strategy in which a ligand is immobilized on a solid support through chemical modifications and is used to capture a functional receptor through ligand–receptor interactions.

Vapour diffusion crystallography

The most commonly used crystallization method for proteins in which a drop of purified protein solution in buffer and precipitant is equilibrated against a higher concentration of precipitant in a larger reservoir. During the equilibrium process, as the concentration of protein and precipitant increases in the crystallization drop, crystals grow depending on the suitability of the condition.

Soaking experiments

In the context of protein crystallization, soaking experiments refer to the incubation of pre-formed crystals with their ligands, for example, an inhibitor. This method is primarily used to obtain crystal structures of apo (ligand-free) and ligand-bound protein.

Biased signalling

G protein-coupled receptors can signal through two parallel and independent pathways: the G protein-dependent and the β-arrestin-dependent pathways. When a receptor signals preferentially through one of these pathways, it is referred to as biased signalling.

Hydrogen–deuterium exchange mass spectrometry

This technique — in which deuterium in solution is exchanged with the backbone amide hydrogen — is used to study conformational changes and dynamics of proteins. The extent and rate of this exchange, measured by mass spectrometry, reflects the local and overall conformational flexibility and dynamics of the protein. This technique has been used to study the conformational dynamics of the agonist–β2-adrenoceptor–G protein complex and the agonist–β2-adrenoceptor–β-arrestin 1 complex.

Combinatorial biology

An approach in which a large number of variants (for example, of a peptide or protein) are generated as a library and screened to find a variant that binds to the target with high affinity. Phage display is a commonly used combinatorial biology approach.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ghosh, E., Kumari, P., Jaiman, D. et al. Methodological advances: the unsung heroes of the GPCR structural revolution. Nat Rev Mol Cell Biol 16, 69–81 (2015). https://doi.org/10.1038/nrm3933

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrm3933

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing